Biogenic silver/silver chloride nanoparticles inhibit human cancer cells proliferation in vitro and Ehrlich ascites carcinoma cells growth in vivo

Silver/silver chloride nanoparticles (Ag/AgCl-NPs) were synthesized for the first time from the herbal Geodorum densiflorum rhizome extracts and characterized by different techniques. The surface plasmon resonance peak at 455 nm was observed in the UV–Visible spectrum, the average particle size of 25 nm was determined by SEM, XRD reflection peaks (28.00°, 32.42°, 38.28°, 46.38°, 54.94°, 57.60°, 64.64°, and 67.48°) indicated the presence of Ag-NPs and AgCl-NPs, heat stability was confirmed by TGA and FTIR analysis indicated the presence of alcohol/phenol, alkanes, primary amines, nitro compounds, alkyl chloride functional groups. The synthesized Ag/AgCl-NPs, previously synthesized Kaempferia rotunda and Zizyphus mauritiana mediated Ag/AgCl-NPs separately inhibited the proliferation of BxPC-3 cells with the IC50 values of 7.8, 17.1, and 20.1 µg/ml, respectively. In the case of MCF-7 cells, the IC50 values of G. densiflorum- Ag/AgCl-NPs and K. rotunda-Ag/AgCl-NPs were 21.5 and 23.5 µg/ml, respectively. Whereas the IC50 of G. densiflorum-Ag/AgCl-NPs was 28.0 µg/ml against glioblastoma stem cells (GSCs). Induction of apoptosis in GSCs, BxPC-3 and MCF-7 cells was noted followed by NPs treatment. In GSCs, the expression level of NFκB, TNFα, p21, and TLR9 genes were upregulated after treatment with G. densiflorum-Ag/AgCl-NPs while in the MCF-7 cells, the expression of p53, FAS, Caspase-8 and -9, NFκB, MAPK, JNK and p21 genes were increased. G. densiflorum-Ag/AgCl-NPs inhibited 60% and 95% of EAC cells growth at the doses of 2 and 4 mg/Kg/day after intraperitoneal treatment with five consequent days, respectively. A remarkable improvement of hematological parameters with the decreased average tumor weight and increase of 75% life span of G. densiflorum-Ag/AgCl-NPs treated mice were observed. Altogether, this study reported for the first time in vitro anticancer activity of biogenic G. densiflorum-Ag/AgCl-NPs against GSC cells along with MCF-7 and BxPC-3 cells and in vivo anticancer properties against EAC cells.


Identification of caspase-3 protein expression in GSCs by the immunofluorescence assay.
To detect the expression level of caspase-3 in GSCs, 2 × 10 4 cells were seeded in each well of a 96 wells plate then the cells were treated with 16 μg/ml of G. densiflorum-Ag/AgCl-NPs for 48 h. Then the expression level of caspase-3 protein was detected by fluorescence microscope followed by the treatment with 4% paraformaldehyde, 0.1% Triton X-100, phosphate buffer saline, 10% goat serum, 0.5% tween-20, and 1% BSA in PBS and finally, caspase-3 primary antibody and Cy3 goat-anti Rabbit IgG antibody (Life technology) followed by the protocol described by Kabir et al. 1 . Changes of genes expressions in GSCs and MCF-7 cells. 16 TCC CGG TCC AGC TAT   R CAC GTC CAA CTC ACT CCA AGG   TNFα  F ATT GCC GCA GAA AGT TCT ACG   R GTC CAG TTT CGT CTT CAG CTC   18S  F GTA ACC CGT TGA ACC CCA TT   R CCA TCC AAT CGG TAG TAG CG   TLR9  F CTG CCT TCC TAC CCT GTG AG   R GGA TGC GGT TGG AGG ACA A   NOTCH2  F CAA CCG CAA TGG AGG CTA TG   R GCG AAG GCA CAA TCA TCA ATGTT   p53  F GCC CAA CAA CAC CAG CTC CT   R CCT GGG CAT CCT TGA GTT CC   P21  F TGC AAC TAC TAC AGA AAC  housed in cages (6 mice/cage) with free access to food and water. All animals were kept under a 12-h/12-h light/ dark cycle (lights on at 6:00 a.m.). The mice were sacrificed in the current study using a two-step process as per the approved guideline. Firstly, the mice were rendered unconscious through inhaled anesthetic agent (Isoflurane) exposure. Subsequently, they were killed by cervical dislocation while the animals were fully unconscious. Isoflurane was administrated by drop method in a container with a tightly fitted lid.

Staining of MCF-7 and BxPC-3 cells with the FITC labeled annexin
In vivo EAC cell growth inhibition. EAC cells propagated intraperitoneally in the ambient of our departmental laboratory biweekly. EAC cells were collected in saline from Swiss albino mice bearing 6 or 7 days old ascites tumors and then 1 × 10 6 cells in 0.1 mL of saline were injected intraperitoneally to 18 Swiss albino mice and incubated at room temperature for tumor inoculation. 24 h later, the mice were randomly distributed into three groups (six mice per group) and G. densiflorum-AgCl-NPs were injected intraperitoneally into two groups at the doses of 2.0 and 4.0 mg/kg/day for five consecutive days. The rest of the group was used as a control. The mice sacrificed on the seventh day of the EAC inoculation and EAC cells were collected in normal saline. Finally, cells were counted by a light microscope and the percent of cell growth inhibition was calculated.

Determination of average tumor weight and survival time.
Twelve Swiss albino mice were treated with EAC cells as described above and after 24 h one group (six mice per group) was treated with 4.0 mg/kg/ day of G. densiflorum-AgCl-NPs for ten consecutive days. The NPs untreated group was used as a control. The third group of mice without EAC and nanoparticles was used as normal mice. After treatment with G. densiflorum-AgCl-NPs, changes in the weight of each mouse were recorded daily up to 20 days of EAC cells inoculation and the host survival time was recorded. Finally, the mean survival time (in days) and the percent increase of life span were calculated as described by Kabir et al. 24 .
Hematological parameters of normal, EAC inoculated and G. densiflorum-AgCl-NPs treated on EAC inoculated mice. Mice that were used for the determination of average tumor weight and survival time were also subjected to the study of hematological parameters. About 50 µl of blood was drawn from the tail of each mouse of the EAC bearing control, G. densiflorum-AgCl-NPs treated EAC bearing and normal mice on the 12th day of EAC cells inoculation. Finally, the percentage of hemoglobin, total WBC and total RBC were counted.

Synthesis of G. densiflorum-Ag/AgCl-NPs and the morphological characterization.
The deepest brown color solution was obtained after incubation with 4.0 mM of silver nitrate that preliminary supported the formation of G. densiflorum-Ag/AgCl-NPs. The maximum absorbance peak in the UV-visible spectra was observed near 455 nm (Fig. 1A). About 215 mg (15 mg/ml) of Ag/AgCl-NPs was obtained from 100 g of G. densiflorum rhizome. The picture of SEM indicated the synthesized Ag/AgCl-NPs were spherical and the average size was 25 nm ( Fig. 1B and C).   Fig. 4B and C, respectively. ROS was generated in MCF-7 cells after treatment with K. rotunda-Ag/AgCl-NPs, while no ROS was detected in control and G. densiflorum-Ag/AgCl-NPs (Fig. 4D).

Changes of gene expression in the treated GSCs and MCF-7 cells. After incubation of GSCs with
the G. densiflorum-Ag/AgCl-NPs, the expression level of NFκB, TNFα, p21 and TLR9 increased while almost no change was observed in the NOTCH2 gene (Fig. 5A). The expression level of caspase-8, NFκB, MAPK and p21 were increased significantly while p53, caspase-9, and FAS expression increased a little and no big alteration was found for TNFα, in MCF-7 after treatment with G. densiflorum-Ag/AgCl-NPs (Fig. 5B).

EAC cells growth inhibition and hematological parameters. After treatment with G. densiflorum-
Ag/AgCl-NPs, EAC cells growth was inhibited effectively. At the dose of 2 mg/Kg/day, cell growth inhibition was 60% and the growth inhibition increased to 95% at the dose of 4 mg/Kg/day as shown in Fig. 6A  www.nature.com/scientificreports/ AgCl-NPs treated mice. Total RBC of normal and G. densiflorum-Ag/AgCl-NPs treated EAC bearing mice were very close when compared with the EAC bearing control mice (Fig. 6B). A remarkable decrease in WBC was observed after treatment of EAC-bearing mice with G. densiflorum-Ag/AgCl-NPs (Fig. 6C). The Hemoglobin level increased after treatment of EAC-bearing mice with the G. densiflorum-Ag/AgCl-NPs (6D).

Effects of G. densiflorum-Ag/AgCl-NPs on average tumor growth and the mean survival time of EAC Cells bearing mice.
EAC-bear mice were treated with G. densiflorum-Ag/AgCl-NPs and 20 days later, the average tumor growth in mice was remarkably low as compared with the EAC-bearing control mice (Fig. 7A). The life span of the G. densiflorum-Ag/AgCl-NPs treated mice was increased by 75% as compared to EAC-bearing control mice (Fig. 7B).

Discussion
For the treatment of cancer, scientists are finding different therapeutic agents those stimulates apoptosis in the cancer cell. The apoptotic inducers derived from phytochemicals have attracted much attention to researchers due to the less toxic effects than those of radiation and chemically synthesized chemotherapeutic agents. Synthesis of nanoparticles by treating AgNO 3 with such type of extract attracted several cancer researchers. For this reason, we have synthesized some nanoparticles which showed cytotoxicity against different cancer cell lines 1,2,17 . In the present study G. densiflorum rhizome was used for the first time for the synthesis of Ag/AgCl-NPs by treating with AgNO 3. After synthesis of Ag/AgCl-NPs the color of the extract was changed from transparent to deep  Several studies showed that biogenic silver nanoparticles retain the ability to inhibit cancer cell growth in vitro and also in vivo in mice and the ability to inhibit cancer cell growth varies on the source of the synthesized silver nanoparticles 1,2,19,[27][28][29][30][31][32] . In the present investigation, we have examined the anticancer activity of the G. densiflorum-Ag/AgCl-NPs, K. rotunda-Ag/AgCl-NPs and Z. mauritiana-Ag/AgCl-NPs against BxPC-3 cells for the first time for any biogenic silver nanoparticles. The IC 50 values indicated G. densiflorum-Ag/AgCl-NPs were the most toxic against BxPC-3 cells. Then the mechanism of anticancer activity of G. densiflorum-Ag/ AgCl-NPs and K. rotunda-Ag/AgCl-NPs against BxPC-3 was studied using FITC-annexin V/PI assay. Both of the nanoparticles inhibited cell growth by the induction of early and late apoptosis.
Anticancer activity of the G. densiflorum-Ag/AgCl-NPs was also checked against GSCs cells and inhibition of the growth of the cells was observed with the IC 50 value of 28.0 µg/ml. Recently, it was reported that K. rotunda-Ag/AgCl-NPs and Z. mauritiana-Ag/AgCl-NPs also inhibited the GSCs with the IC 50 values of 6.8 and That demonstrated the newly synthesized nanoparticles were less effective against GSCs. Cell growth inhibition was due to the induction of apoptosis which was confirmed by the immunofluorescence study of caspase-3 protein expressions. Similar results were also obtained in our early experiments of the K. rotunda-Ag/ AgCl-NPs and Z. mauritiana-Ag/AgCl-NPs against GSCs 1 . Here the expression level of TLR9, p21, TNFα and NFκB genes increased and almost no change was observed for NOTCH2 gene that is responsible for self-renewal, cell proliferation and cell maintenance of GSCs. Our previous study demonstrated that K. rotunda-Ag/AgCl-NPs inhibited the GSCs by increasing the TLR9, p21, TNFα and NFκB genes with the several folds decrease of NOTCH2 1 . Like our early report, the present study also illustrated that G. densiflorum-Ag/AgCl-NPs activated TLR9 gene that activated NFκB sequentially by activating TNFα and, finally, NFκB entered the nucleus and broke the DNA, resulting in induction of apoptosis in GSCs cells. Opposite result was also reported by Akter et al. 33 46 . Our present study stated the involvement of various genes in the apoptosis process of the MCF-7.
In vitro anticancer study using biogenic Ag/AgCl-NPs is common but only view literature stated the in vivo study 9,[12][13][14] . In our previous study, we found that Z mauritiana-Ag/AgCl-NPs and K rotunda-Ag/AgCl-NPs inhibited 20% and 55% of EAC cells growth inhibition at the dose of 12 mg/Kg/day dose, respectively. While the dose was decreased to 6 mg/Kg/day no cell growth was observed for Z mauritiana-Ag/AgCl-NPs and 32.3% of EAC cells growth inhibition was observed for K rotunda-Ag/AgCl-NPs 1,2 . However, in the present study, 95% and 60% of cells growth inhibition were observed at 4 and 2 mg/Kg/day doses, respectively. The result indicated the G. densiflorum-Ag/AgCl-NPs is more active than that Z mauritiana-Ag/AgCl-NPs and K rotunda-Ag/AgCl-NPs.
Besides the EAC cells growth inhibition, for the first time, we are reporting the result of the investigation of the hematological parameters, average tumor growth and life span. After treatment of biogenic silver nanoparticles, the total RBC and hemoglobin level in the G. densiflorum-Ag/AgCl-NPs treated EAC bearing mice increased with the decrease of WBC. Typically, anemia occurs in the EAC-bearing mice due to the decrease of RBC and percent of hemoglobin. After treatment with the nanoparticles, RBC and hemoglobin levels were increased and the infection was reduced as the WBC was decreased. In the EAC-bearing mice, a rapid increase of ascites fluid is observed. The ascites fluid is the direct nutritional source of EAC cells. After treatment with G. densiflorum-Ag/ AgCl-NPs, the volume of ascites fluid and the number of EAC cells decreased with the increased life span. These results indicated the synthesized nanoparticles would be a candidate for strong anticancer agents.

Conclusion
In conclusion, G. densiflorum-Ag/AgCl-NPs were synthesized and characterized that retained the anticancer activity against BxPC-3, GSCs and MCF-7 cells. Although the anticancer activity of the G. densiflorum-Ag/AgCl-NPs against BxPC-3 and MCF-7 was stronger than K. rotunda-Ag/AgCl-NPs and Z. mauritiana-Ag/AgCl-NPs, however, weaker than those nanoparticles against GSCs. The G. densiflorum-Ag/AgCl-NPs might be a strong anticancer agent against the BxPC-3 and MCF-7 cells. In vivo experiments indicated that G. densiflorum-Ag/ AgCl-NPs can be a promising anticancer agent and can be used for further investigations.
Statistical analysis. The experimental results have been expressed as the mean ± SD (Standard Deviation).
Data have been calculated by the one-way ANOVA followed by the Dunnett 't' test using SPSS software of version 16.